Fig. 3 |. Constructing the UCI-ORF dictionary.

a, (i) Tagmentation randomly inserts adapters into the MIPSA vector library. (ii) Using a PCR1 forward primer and the reverse primer of the tagmentation-inserted adapter, DNA fragments are amplified and size selected to be ~1.5 kb, which captures the 5’ terminus of the ORF. (iii) These fragments are amplified with a P5-containing PCR2 forward primer and a P7 reverse primer. (iv) Illumina sequencing is used to read the UCI and the ORF from the same fragment, thus enabling their association in the dictionary. b, The number of monospecific UCIs is shown for each member of pDEST-MIPSA hORFeome library, superimposed on the length of the ORFs. c, Histogram of ORF representations in the library according to their aggregated UCI-associated read counts. Vertical red lines show +/−10x the median UCI-associated read count. d, IP of hORFeome MIPSA library using Sjogren’s Syndrome (SS) plasma is compared to the average of 8 mock IPs. Sequencing read count for each UCI are plotted. UCIs associated with the two GAPDH isoforms (filled black) and spiked-in TRIM21 (red) are indicated